Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression . Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI:  Mammary gland stem cells are capable of self-renewal and to generate all differentiated progeny of the gland , 2. Becasrc transfection leads to increased expression of glucose uptake . Cell lysates were obtained by centrifugation at 21,380xg for 30 min at 4°C; protein concentration in the supernatant was determined by BCA protein assay , and lysates were adjusted to equivalent concentrations with lysis buffer. Aliquots of 10–40 μg of total cell lysate were then separated on SDS–PAGE. Proteins were transferred to PVDF membranes that were blocked overnight at 4°C with 5% non-fat milk in TTBS (TBS with 0.05% Tween-20). Incubation with primary specific antibodies was carried out overnight at 4°C, and horseradish peroxidase-conjugated secondary antibodies in blocking solution for 1 h at room temperature. Immunoreactive bands were visualized by ECL kit.Preparation of cell lysates from mammospheres and immunoblotting analyses were carried out as previously described . Brieflyrd generation from two independent experiments cultured either in the absence (Control) or presence of Doxy (2 μg/mL) were collected by centrifugation, washed with PBS, and lysed in 9 M urea, 2 M thiourea, 5% CHAPS, and 2 mM TCEP. The samples were precipitated by methanol/chloroform and quantified by Pierce 660nm Protein Assay. Then 30 μg of protein from each sample were trypsinized, and the resulting peptides were labeled using iTRAQ 4-plex reagent  according to the manufacturer's instructions and named as follows: Control-1, 114-tag; Doxy-1, 115-tag; Control-2, 116-tag; Doxy-2, 117-tag. Samples were pooled, evaporated to dryness and frozen. The pool was separated into 5 fractions in a reverse phase C18 chromatography at basic pH and cleaned with a StageTip-C18. The 5 fractions were quantified by fluorimetry, and 1 μg of each fraction was separated by C-18 reverse phase chromatography during 150 min. The eluted peptides passed then into 5600 Triple-TOF MS . The raw data of the combined 5 fractions was exported to the search-engine Mascot Server v2.5.1  confronted to the Homo sapiens UniProt database. Peptide mass tolerance was set to ± 25 ppm for precursors and 0.05 Da for fragment masses. Frequency distribution histograms of protein ratios were obtained into Excel 2010. Log2 peptide ratios followed a normal distribution that was fitted using least squares regression. Mean and standard deviation values derived from the Gaussian fit were used to calculate p-values and quantification of False Discovery Rates (FDR). The confidence interval for protein identification was set to ≥ 95% (p<0.05) and only peptides with an individual ion score above the 1% False Discovery Rates (FDR) threshold were considered correctly identified. Only proteins having at least two quantitated peptides were considered in the quantitation. A 5% quantitation FDR threshold was estimated to consider the significant differentially expressed proteins.Briefly, MCF7-Tet-On-SrcDN derived mammospheres at 310.6019/PXD017789).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner rd generation derived from MCF7-TET-ON-SrcDN cells were seeded into a six-well plate and cultured with fresh medium -/+ Doxy (2 μg/mL) for 12–24 h. Cells were harvested by centrifugation, washed twice with PBS and preincubated for 10 min in PBS. Then, cells were collected by centrifugation, suspended in fresh culture medium and incubated for another 4 h. Cells were harvested by centrifugation at room temperature at 0 and 4 h incubation and treated with 200 μL lysis buffer (Tris-HCl (pH 7.5), 1 mM EDTA, 0.5 mM dithiothreitol, 0.2% Triton X-100, 0.2 mg/mL deoxycholate, 0.2 mM phenylmethylsulfonyl fluoride, and 2.5 μg/mL leupeptin), and homogenized at 4°C by pipetting up and down. The homogenate was centrifuged at 21,380xg for 15 min at 4°C and the supernatant was used for enzymatic activity measurements. Hexokinase (HK) activity was determined spectrophotometrically at 340 nm by following the G6PDH dependent conversion of NADP to NADPH. The assay mixture contained 100 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 10 mM MgCl2, 5 mM MgATP, 2 mM glucose, 0.5 mM NADP+ and 10 U/mL glucose 6-phosphate dehydrogenase (G6PDH) in a final volume of 1 mL. HK activities were calculated according to the slope of the resulting curves in the log phase. The initial velocity of lactate dehydrogenase (LDH) was determined by measuring spectrophotometrically the NADH consumption at 340 nm in a 1 mL reaction mixture . The slopes of the initial linear decrease were used to calculate initial velocities. Pyruvate kinase (PK) activity was measured by following the production of pyruvate. The reaction mixture consisted of 50 mM HEPES (pH 7.2), 100 mM KCl, 5 mM MgCl2, 0.15 mM NADH, 1 mM MgADP, 2 mM phosphoenolpyruvate, and 1 U/mL of LDH as the auxiliary enzyme in a final volume of 1 mL. PK activity was determined spectrophotometrically as above by recording the change of 340 nm absorbance produced by NADH oxidation. One unit of enzyme activity is defined as the amount of enzyme catalyzing the conversion of 1 μmol of substrate/min at 25°C. Enzymatic activities of different samples were normalized against the corresponding protein concentrations in the lysates.Mammospheres at 3g for 15 min at 4°C, the supernatant was neutralized to pH 7.0 with a 2 M KOH and 0.5 M triethanolamine solution, the mixture was incubated on ice for 15 min, and precipitated KClO4 was removed by centrifugation. Glucose and lactate content in culture medium and levels of intracellular lactate were assessed by using standard enzymatic techniques Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: No**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/AReviewer #2: I Don't Know**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: No**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have previously constructed a line of MCF7 human breast cancer cells (MCF7-Tet-On-SrcDN cells) in which expression of a dominant-negative form of the avian c-Src protein tyrosine kinase (SrcDN) can be induced by doxycycline (DOX) treatment, and shown that SrcDN expression inhibited cell attachment, spreading, and migration and also reduced tumorigenesis. Here, they have extended their studies of the phenotypic consequences of inducibly expressed SrcDN, and examined the effect SrcDN expression on the stem cell-like properties of a population of sorted CD44-high/CD24-low cells (BCSCs) MCF7-Tet-On-SrcDN cells that can form mammospheres in culture and initiate tumor growth in vivo. They found that induction of SrcDN expression in mammospheres increased the level of pY397 FAK, as reported previously, but that the level of E-cadherin was unaffected. They went on to show that mammosphere formation efficiency was reduced ~40% by the third passage of CD44-high/CD24-low cells when SrcDN was induced, suggesting that Src function is needed for BCSC self-renewal. To define the effect of SrcDN induction on BCSCs, immunoblotting for proteins of interest and a global proteomic analysis was carried on control and SrcDN expressing mammospheres. Decreases in cyclin D1, Nanog, Oct4 and ALDH1 were noted, consistent with reduced stemness. The global proteomic analysis revealed decreased levels of 15 proteins, with proteins involved in glycolysis being prominent. Next they tested whether SrcDN expression affected glycolysis, and found reduced glucose uptake and lactate excretion, paralleling a reduction in GLUT1, HK2 and LDHA levels, and decreased hexokinase, pyruvate kinase and lactate dehydrogenase enzymatic activity, all consistent with a reduced glycolytic rate. They also found that there were reduced levels of MCT1, the lactate transporter, explaining the observed rise in the level of intracellular lactate. Finally, they showed that cells dissociated from SrcDN-expressing mammospheres had altered maximum oxygen consumption rates in cells cultured in pyruvate-containing medium, albeit without a significant effect on ATP levels.The observed effects of SrcDN expression on the stemness and metabolic properties of the CD44-high/CD24-low population of stem cell-like cells isolated from cultured MCF7-SrcDN cells are quite interesting, and suggest that that Src family kinase activity is needed to maintain the stemness state and glycolytic properties of BCSC cells. The data are largely convincing, and imply an important role for Src famliy kinase signaling in maintaining a stem cell-like population, although no mechanistic insights are presented into how Src kinase activity results in the observed changes in stem cell and metabolic genes.Points: 1. Figure 1: The CD44-high/CD24-low population of BCSCs was isolated from MCF7-Tet-On-SrcDN cultures without induction of SrcDN, and then cells were plated to form mammospheres. However, it is not clear from the legend how long the mammospheres, once formed, were treated with DOX prior to the immunoblotting analysis or what happened to the acinar structures during the DOX treatment period, e.g., do they maintain their morphology. It is also not clear what happens to the phenotype of BCSC cells once a mammosphere has been formed - do all the cells retain their BCSC phenotype or have some of them differentiated back into CD44-high/CD24-high cells. The authors report that when mammospheres were dissociated into single cells and then re-plated they could form mammospheres, but information about the efficiency of this process was not provided. If the dissociated cells are re-analyzed by FACS, what fraction are CD44-high/CD24-low and what fraction are CD44-high/CD24-high. If the cells are treated with DOX either prior to (i.e. as mammospheres) or during the re-plating process with DOX, how does this affect the efficiency of mammosphere formation (see point 2)?2. Figure 2A: It is not clear from the legend or text exactly what was done in this experiment. Were the single cells that were plated obtained from mammospheres that had been pretreated with DOX, or were the cells continuously treated with DOX during plating and mammosphere outgrowth? This needs to be spelled out in the legend.3. Figure 2B: How long was SrcDN induced for in the mammospheres prior to harvesting for the proteomic analyses?4. Table 1: It is unclear from the text and the supplementary materials how many total proteins were identified through the MS analysis, i.e. the 15 protein decreases and 4 protein increases need to have a denominator of the total number of proteins identified. By comparative immunoblotting of control and DOX-induced MCF7-SrcDN mammospheres, the authors showed that the levels of several proteins involved in stem cell function and the glycolytic pathway were decreased upon SrcDN induction. However, it is not clear how many of these proteins were also found to be downregulated in their proteomic dataset - none of the proteins tested by immunoblotting is listed in Table S1, but I did not check the data deposited in the ProteomeXchange.5. Figure 2: Have the authors carried out a parallel RNA-seq analysis on control and DOX-induced MCF7-SrcDN cells to determine which of the observed changes in protein levels can be attributed to decreased levels of the cognate mRNAs . It would be particularly interesting to know whether SrcDN expression affects the levels of Myc and HIF1 RNA expression, since these transcription factors are known to promote expression of the HK2 and LDHA genes. Also, are there any changes in the levels of expression of RNAs encoding mitochondrial ETC genes? Likewise, analysis of the differences in RNA expression patterns between CD44-high/CD24-low BCSCs and the CD44-high/CD24-high cells would be informative.6. Figure 3: Although an ~50% decrease in pY10 LDHA level upon DOX induction of SrcDN is reported in panel D, there is a concern about how accurate this value is - the low level of the pY10 immunoblot signal means that the percent decrease will be very dependent on how accurately the background signal, which needs to be subtracted, was estimated. A bigger decrease than the observed 50% in pY10 LDHA level might have been expected given the very high level of SrcDN expression compared to that of endogenous c-Src in the induced cells. In this regard, it has been reported (ref. 18) that the level of pY10 in MCF7 cells is very low compared to that in other more advanced breast cancer lines.Minor points: 1. The first paragraph of the results is entitled “SrcDN reduced self-renewal of MCF7-BCSCs” but no such data are described in this section.2. While many readers may know what MCF7 cells are, the authors should describe them in the Introduction, i.e. they are ER/PR-positive, HER2-negative, luminal A-type, non-metastatic human breast cancer cells.3. Do the cellular phenotypes of DOX treated BCSCs revert when DOX is withdrawn, and if so how rapidly?4. Page 12, second paragraph: The authors say that the endogenous c-Src cannot be distinguished from the exogenously expressed SrcDN by proteomic analysis, but this is not actually true, since they expressed chicken c-SrcDN in human cells, and there are a significant number of sequence differences between the two c-Src’s, which would mean that the chicken and human c-Src proteins would generate unique peptides that could be quantified.5. Although technically more challenging because of the small cell numbers, it would have been interesting to analyze the sorted CD44-high/CD24-low BCSC MCF7 cell population directly for changes in protein levels, glycolysis rates, etc, compared to the CD44-high/CD24-high population.Reviewer #2: In this study, the authors tried to investigate whether SrcDN reduces tumorigenesis of MCF7 cells via inhibiting breast cancer stem cells. They isolated BCSCs and non-BCSCs based on ESA, CD44, and CD24 markers and evaluated the effect of SrcDN on the metabolic changes. Though somewhat interesting, the data are underdeveloped and not supporting the conclusion. The major flaw is that they didn’t show that the changes in glycolysis and decrease in self-renewal were unique to the BCSCs. Except isolating these different cell populations and analysis of the E-Cad levels, there are no experiments done comparing these two groups. Without such analysis, the conclusion of the SrcDN on BSCS to inhibit tumorigenesis is not supported. There were several syntax/grammatical errors throughout the manuscript, which should be improved.In addition, they should have performed direct analysis to address whether the SrcDN induced glycolysis changes are directly connected to self-renewal properties, since they showed it wasn’t related to ATP production or ROS.Specific comments:Figure 1:1. Figure 1A, isotype control for setting up the gate for the FACS should be included.2. Figure 1B, it is unclear the significance of this figure, since there is no comparison of spheres formed by CD24- and CD24+ groups. There is lack of scale bars.3. Other methods have been used to isolate putative BCSCs, particularly using ALDH activity for the luminal breast cancers. To strengthen the claim on BCSCs, the authors should also consider using different ways to evaluate BCSCs.Figure 2:1. Figure 2A, possibly due to sample size or heterogeneity in the mammospheres, without seeing the full error bars and with the statistical significance being relatively large, this data is not very strong. It is also unclear what the bar and the error bar show. mean or median? SD or SEM?2. In Figure 2B, the quantitation for the Nanog and Oct3/4 Western blots were the same, but the blot for Oct3/4 was far less apparent compared to Nanog, especially if there were compared to the same β-Actin loading control.Figure 3:1. A-C. As in Figure 2, the lack of full error bars and small sample size makes the data less convincing, especially because each graph has the same inferred statistical significance from very different measurements. Again, it is unclear what the error bars represent.2. In Figure 3D, The blots for Glut-1 and pY10-LDHA, and LDHA are not as well defined as the others, although it could be due to the antibodies. The quantitative decreases in Glut-1, HK2, and pY10-LDHA/LDHA are not very significant and it is unclear whether such level of a decrease can cause changes in glycolytic activity/output. There are no functional validation or rescue experiments done to link the protein level changes to the glycolysis changes.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  9 Jun 2020We have elaborated a large document that we named "Reviewers_Reply_03-06-2020" in PDF format where we have answered the questions raised by the Reviewers and the Editor. We must say that we are thankful to the comments raised, as we think that they help us to improve the quality of the manuscript.AttachmentReviewers_Repply_03-06-2020.pdfSubmitted filename: Click here for additional data file. 24 Jun 2020c-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cellsPONE-D-20-06055R1Dear Dr. Martín-Pérez,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Antimo Migliaccio, M.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have satisfactorily addressed most of my comments, and have added some new data as supplementary figures, including Figure S3, where they showed that DOX treatment of MCF7-Tet-On-SrcDN cell-derived mammospheres to induce DN-cSrc expression resulted in a strong decrease in the numbers of CD44-high/CD24-low stem cell-like cells, which have high tumorigenicity, and a corresponding increase in the number of CD44-high/CD24-high cells, which show reduced tumorigenicity, consistent with the observed DOX-induced decrease in stem cell factors such as Nanog.Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: Yes: Tony HunterReviewer #2: No 30 Jun 2020PONE-D-20-06055R1 c-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cells Dear Dr. Martín-Pérez:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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We attempted to design an ophthalmic in situ gel formulation incorporating disulfiram (DIS) nanoparticles (Dis-NPs/ISG) and demonstrated the therapeutic effect of Dis-NPs/ISG on retinal dysfunction in 15-month-old Otsuka Long–Evans Tokushima Fatty (OLETF) rats, a rat model of diabetes. The DIS particles were crushed using a bead mill to prepare the nanoparticles, and the Dis-NPs/ISG was prepared using a combination of the DIS nanoparticles and an in situ gelling system based on methylcellulose (MC). The particle size of the Dis-NPs/ISG was 80–250 nm, and there was no detectable precipitation or aggregation for 1 month. Moreover, the Dis-NPs/ISG was gelled at 37 °C, and the drug was delivered into the retina by instillation. Only diethyldithiocarbamate (DDC) was detected in the retina (DIS was not detected) when the Dis-NPs/ISG was instilled in the right eye, and the DDC levels in the right retina were significantly higher than those in the left retina. In addition, the retinal residence time of the drug was prolonged by the application of the in situ gelling system, since the DDC levels in the retinas of rats instilled with Dis-NPs/ISG were higher than those in DIS nanoparticles without MC. Furthermore, repetitive instillation of the Dis-NPs/ISG attenuated the deterioration of electroretinograms (ERGs) in 15-month-old OLETF rats by preventing the collapse of ATP production via excessive nitric oxide and recovered the decrease in retinal function. These findings provide important information for the development of novel therapeutic approaches to diabetic retinopathy. It is predicted that the proportion of patients with diabetes mellitus (DM) will increase to 4.4% in 2030, and the management and prevention of DM are now global public health problems ,2. DM paAkita mice, a mouse model of type 1 diabetes, and rats with streptozocin-induced diabetes (STZ rats) have been used as animal models to investigate DR [The pathogenic mechanisms of DR were investigated using animal models of DM, and Ins2igate DR ,7. Howevigate DR ,10,11,12igate DR ,14. The igate DR . Thus, Oigate DR . Then, tigate DR .Disulfiram (DIS) is a dimer of diethyldithiocarbamate (DDC) and has long been used to treat alcoholic syndrome. The DIS is a potent NOS inhibitor and a radical scavenger, and our previously studies showed that the converted DDC, after the oral administration of DIS, attenuated the iNOS in the retina and prevented the collapse of ATP production in the retinas of 15-month-old OELTF rats . FurtherBA), we attempted to combine nanoparticles and an in situ gelling system. We also reported that a combination of nanoparticles and an in situ gelling system using methylcellulose (MC) increased the ocular BA [BAs may enable drug delivery to the retina.The blood–retinal barrier, corneal barrier, and tear film barrier restrict the entry of drugs into the eye. Due to the structure and complex physiology of the eye, it is much harder to deliver drugs to the posterior segment , and inscular BA ,27. ThesIn this study, we attempted to design an ophthalmic in situ gel formulation incorporating DIS nanoparticles (Dis-NPs/ISG) and investigated whether the Dis-NPs/ISG delivered the drug into the retina by instillation. Moreover, we demonstrated the therapeutic effect of Dis-NPs/ISG on retinal dysfunction in 15-month-old OLETF rats.Male 15-month-old control Long–Evans Tokushima Otsuka rats  and OLETF rats were obtained from Hoshino Laboratory Animals, Inc. These animals were housed at 25 °C (room temperature) and freely fed a commercial diet  and water. All procedures were approved by Kindai University on 1 April 2013 (project identification code KAPS-25-003) and were performed in accordance with the guidelines of the Association for Research in Vision and Ophthalmology on the use of animals in research.Isoflurane, benzophenone, mannitol (D-mannitol), a Cholesterol E-Test Kit, and DDC were purchased from Wako Pure Chemical Industries, Ltd. . DIS powder and 2-hydroxypropyl-β-cyclodextrin (HPCD) were kindly donated by Ouchi Shinko Chemical Industrial Co., Ltd.  and Nihon Shokuhin Kako Co., Ltd. , respectively. A Mitochondrial Isolation kit, a Cytochrome c Oxidase Assay kit, and an ATP Bioluminescent Assay kit were purchased from Sigma Aldrich Japan . A Bio-Rad Protein Assay kit was provided by BIO-RAD . We obtained 0.5% phenylephrine and 0.5% tropicamide from Santen Pharmaceutical Co., Ltd. . An ELISA Insulin Kit and an RNA PCR Kit (AMV Ver 3.0) were purchased from Morinaga Institute of Biological Science Inc.  and Takara Bio Inc. , respectively. Methylcellulose type SM-4 (MC) was obtained from Shin-Etsu Chemical Co., Ltd. . Pentobarbital was provided by Sumitomo Dainippon Pharma Co., Ltd. . Benzalkonium chloride (BAC), methanol, and acetonitrile was purchased from Kanto Chemical Co., Inc. . LightCycler FastStart DNA Master SYBR Green I was provided by Roche Diagnostics Applied Science . All other used chemicals were of the highest purity commercially available.One percent DIS powder was added to a 10% HPCD solution containing 0.1% mannitol and 0.001% BAC and stirred for 24 h in the dark at 22 °C. After that, the solution was filtered using a 0.20 µm Minisart CE , and the filtrated solution (the DIS–HPCD inclusion complex) was used as DIS-sol. In the filtrate, the adsorption of DIS was not observed, and the pH was adjusted to 6.5.The mixture containing 1% DIS powder (mean particle size: 97.5 ± 36.4 µm), 0.1% mannitol, 0.001% BAC, and 0.5% MC was placed in a zirconia mortar with a zirconia ball and milled at 100 rpm for 7 d at 4 °C using a stirrer for a magnetic mortar . After that, the mixture was dispersed in purified water, and the dispersions were transferred to a tube with zirconia beads (diameter: 0.1 mm) and milled with the Bead Smash 12  30 times. Then, the milled dispersions with zirconia beads were crushed using a Shake Master NEO  at 1500 rpm and 4 °C for 1 h. The milled dispersions were used as Dis-NPs. Moreover, the Dis-NPs/ISG was prepared by adding 1.5% MC to Dis-NPs. ® ODS-3 column , and the column temperature was set to 35 °C using a CTO-20A column oven . Here, 40% acetonitrile containing 0.1% trifluoroacetic acid was used as a mobile phase and the flow rate was adjusted to 0.25 mL/min, and the levels of DIS and DDC were detected at 215 nm in an SPD-20A UV detector .Fifty microliters of sample was added to 100 µL of methanol containing 0.3 µg of benzophenone  and filtered through a Chromatodisk 4A . Ten microliters of the filtrated solution was injected into an LC-20AT HPLC system using an SIL-10AF auto sampler . We used an InertsilTM MAX-XP Ultracentrifuge . The above-described HPLC method was used to measure the contents of solubilized and unsolubilized DIS and evaluate the solubility. The zeta potential in the DIS formulations was measured using a micro-electrophoresis zeta potential analyzer .The particle size distribution of the DIS powder was measured using a light microscope and an SALD-7100 laser diffraction particle size analyzer . The particle size distribution and the number of DIS nanoparticles were determined using the SALD-7100 and a NanoSight LM10 dynamic light scatterer . Moreover, the number of DIS nanoparticles was also measured using the NanoSight LM10. Atomic force microscope (AFM) images of DIS nanoparticles were provided by an SPM-9700 . A MCR302 rheometer with a CP50-1  was used to measure the viscosities of the DIS at 20 and 37 °C. The unsolubilized DIS in the Dis-NPs and the Dis-NPs/ISG was separated by centrifugation  using a Beckman OptimaThe ophthalmic DIS formulations were stored in the dark at 20 °C for 1 month. Then, solution containing DIS was taken from 5 mm under the surface and used as a sample. The DIS contents in the provided samples were measured by the above-described HPLC method, and the contents were evaluated in terms of the dispersibility of DIS formulations. Moreover, the particle size and number of DIS nanoparticles in the samples were analyzed using the NanoSight LM10 to demonstrate the changes in particle states over time.Thirty microliters of ophthalmic DIS formulation was instilled into the right eye of 15-month-old OLETF rats without anesthesia, and the rats were left for 0–100 min. Then, the rats were killed by injecting a lethal dose of pentobarbital and the retinas were removed. The collected retinas were homogenized in methanol on ice and centrifuged at 20,400 g for 15 min at 4 °C. The DIS and DDC concentrations in the supernatant were measured using the above-described HPLC method.Thirty microliters of Dis-NPs/ISG was repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, the toxicity (wound area) was stained by 1% fluorescein (30 μL). A Topcon TRC-50X  was used to monitor the corneal wound in each rat.Thirty microliters of the vehicle and Dis-NPs/ISG were repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, blood was collected from the tail vein of 15-month-old OLETF rats that had fasted for 12 h without anesthesia. Plasma glucose and triglyceride levels were measured using an Accutrend GCT System . During the measurement of the insulin and total cholesterol levels, the collected blood was centrifuged at 20,400 g for 15 min at 4 °C, and the supernatant was used to measure the plasma insulin and cholesterol levels. The ELISA Insulin Kit and the Cholesterol E-Test Kit were used to analyze the insulin and cholesterol, respectively. All analyses were performed according to the manufacturer’s instructions.260/OD280 values of all RNAs used were greater than 1.8. The RNA PCR Kit was used to perform the RT reaction, and the PCR reactions were performed using LightCycler FastStart DNA Master SYBR Green I. All reactions were performed according to the manufacturer’s instructions. The following primers were used: 5′-CGCTCCTGGAAGATGGTGAT-3′ for glyceraldehyde-3-phosphate dehydrogenase ; 5′-GGAGAGATTTTTCACGACACCC-3′ and 5′-CCATGCATAATTTGGACTTGCA-3′ for iNOS (GenBank accession No. NM_012611); and 5′-ACGGCACAGTCAAGGCTGAGA-3′. The conditions for the PCR were set to 95 °C for 10 min, 50 cycles of 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 5 s. The differences in the threshold cycles for iNOS and GAPDH were used to calculate the levels of mRNA expression in the rats.Thirty microliters of the vehicle and Dis-NPs/ISG were repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, the 15-month-old OLETF rats were killed by injecting a lethal dose of pentobarbital, and the retinas were removed. All of the RNAs were extracted from the retinas using the acid guanidium thiocyanate–phenol–chloroform extraction method. The ODg for 15 min at 4 °C. The supernatants were used to measure NO levels. The NO2− metabolite level is presented as the NO level. The ENO-20 NOx analyzer system was used to measure the NO2. The NO2− was mixed with Griess reagent containing 0.25 g/L N-naphthylethylenediamine, 5 g/L sulfanilamide, and 1.25% HCl and incubated at 35 °C in a column oven. Then, the absorbance of the colored product dye at 540 nm was measured using an NOD-10 spectrophotometer . The NO2− levels are presented as nmol/mg of protein. The Bio-Rad Protein Assay Kit was used to measure the protein levels in the retinas of 15-month-old OLETF rats according to the manufacturer’s instructions.Thirty microliters of the vehicle and Dis-NPs/ISG were repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, the 15-month-old OLETF rats were killed by injecting a lethal dose of pentobarbital, and the retinas were removed. The collected retinas were homogenized in saline on ice, and the homogenates were centrifuged at 20,400 2HPO4, 5 mM sodium succinate, 0.08 mM ADP, 1 mM ATP, 200 mM mannitol, and 10 mM HEPES and used as the isolated mitochondria from retinas. The Cytochrome c Oxidase Assay kit was used to analyze the CCO activity and isolated mitochondria from the retinas of OLETF rats. The analysis was performed according to the manufacturer’s instructions. The CCO activity is presented as units/milligram of protein, and the protein level was measured by the above-described method.Thirty microliters of the vehicle and Dis-NPs/ISG were repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, the 15-month-old OLETF rats were killed by injecting a lethal dose of pentobarbital, and the retinas were removed. The mitochondria in retina were isolated using the Mitochondrial Isolation kit according to the manufacturer’s instructions. The collected retinas were homogenized in isolation buffer containing 2 mg/mL albumin, 1 mM EDTA, 70 mM sucrose, 200 mM mannitol, and 10 mM HEPES. The homogenates were centrifuged at 600 g for 5 min at 4 °C, and the supernatants were centrifuged at 11,000 g for 10 min at 4 °C. The collected pellets were washed with isolation buffer 1. The resulting pellets were vortexed with 40 µL of buffer 2 containing 1 mM DTT, 2 mM KThirty microliters of the vehicle and Dis-NPs/ISG were repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, the 15-month-old OLETF rats were killed by injecting a lethal dose of pentobarbital, and the retinas were removed. The collected retinas were homogenized in 10 mM HEPES/KOH buffer (pH: 7.8) on ice, and the homogenates were centrifuged at 9100 g for 10 min at 4 °C. The supernatants were used to measure ATP levels. The ATP levels were analyzed using the ATP Bioluminescent Assay Kit according to the manufacturer’s instructions. A luciferin–luciferase reaction was detected by an AB-2200 luminometer . The ATP levels are presented as nmol/mg of protein, and the protein level was measured by the above-described method.Thirty microliters of the vehicle and Dis-NPs/ISG were repeatedly instilled into the right eye of OLETF rats (14–15 months of age) for 1 month . Then, the 15-month-old OLETF rats were maintained in a completely dark room for 24 h. Afterward, the pupils in rats anesthetized with isoflurane were dilated by 0.5% phenylephrine and 0.5% tropicamide. ERG readings in the right eye of 15-month-old OLETF rats were performed according to our previous report and recorded by PuREC . The amplitude of the a-wave was measured from the baseline to the maximum a-wave peak, while the b-wave was measured from the maximum a-wave peak to the maximum b-wave peak. The oscillatory potential (OP) amplitudes were measured in the time interval between the a- and b-wave peaks. All procedures were performed under dim red light.t-test was used for statistical comparisons. The significance level was set at p < 0.05. The values are presented as the mean ± standard error of the mean (S.E.M).F test and bartlett’s test were used to assess data normality, and one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test and Student’s BA could be enhanced by the improvement of the corneal permeability time and the precorneal residence of the drug [We previously showed that the instillation of suspensions containing DIS nanoparticles can supply more DDC to the aqueous humor and lens than a conventional formulation (DIS-sol) ,29. Howethe drug . TherefoIt is important to clarify how a drug is delivered to the retina by instillation of ophthalmic DIS formulations. Therefore, the changes in drug levels in the retina were measured. They are shown in OLETF rats are a model of human DM. In this study, we used OLETF rats to evaluate the preventive effect of instillation of the Dis-NPs/ISG on retinal disorders. RD is a retinal disease caused by the adverse effects of hyperglycemia, and, over time, retinal dysfunction leads to a loss of vision. In this study, we designed an ophthalmic in situ gel incorporating DIS nanoparticles (Dis-NP/ISG) and found that the instillation of Dis-NPs/ISG was able to deliver a sufficient amount of drug into the retinas of rats in comparison with traditional ophthalmic formulations. Moreover, we showed that the instillation of Dis-NPs/ISG attenuated the collapse of ATP production by CCO dysfunction via excessive NO and recovered the deterioration of ERGs in the retinas of OLETF rats.DIS is a quaternary ammonium compound and a dimer of DDC. DIS has been used as an anti-alcoholism drug in the clinic, and its long-term use is very safe and well-tolerated with few side effects . After oFirst, we designed ophthalmic DIS formulations. We previously prepared the formulation for the DDC solution, the DIS/HPCD inclusion complex, and the ophthalmic formulation containing DIS nanoparticles (Dis-NPs) ,29 and sThe MC (SM-4) formed a physically cross-linked hydrogel at approximately 32–35 °C. MC is frequently used as a gelling agent in the ophthalmic field , and theThe addition of MC did not affect the particle size and solubility, and the viscosity was enhanced. The viscosity of the Dis-NPs/ISG was 2.85 mPa∙s at 20 °C, the Dis-NPs/ISG gelled at 37 °C, and the viscosity of the Dis-NPs/ISG was 8.01 mPa∙s at 37 °C . FurtherNext, we determined whether the in situ gels incorporating DIS nanoparticles affected the amount of drug delivered into the retina following instillation. Our previous reports using DIS-sol showed that the DIS was converted to DDC by the aldehyde dehydrogenase 3A1 (ALDH3A1), a sulfhydryl-rich protein, in the cornea, and released to the intraocular region . In thisn = 8). These decreases in insulin levels reflect the progression of type 2 DM. Furthermore, the collapse of ATP production via NO was observed in the retinas of 15-month-old OELTF rats, and the a-wave amplitude, the b-wave amplitude, and the OP amplitude in the retinas of OLETF rats were lower in comparison with those in the retinas of normal rats. These results show that low ATP levels may induce the deterioration of ERGs.The selection of the experimental animal is very important to evaluate the therapeutic effect of Dis-NPs/ISG on retinal dysfunction in DM. OLETF rats are an animal model of type 2 DM with a metabolic syndrome and have pathological characteristics resembling those of human type 2 DM patients ,11,12,35In this study, the Dis-NPs/ISG was repeatedly instilled into the retinas of OLETF rats for 1 month (from 14 to 15 months of age), and the repetitive instillation of Dis-NPs/ISG was found to prevent NO production via iNOS mRNA and recover the decrease in ATP and the amplitude of ERGs  in the 15-month-old OLETF rats. The therapeutic effect of retinal ATP dysfunction by instillation of Dis-NP/ISG was similar to that in oral administration of DSF (100 mg/kg body weight) . MoreoveWe succeeded in preparing in situ gelling systems based on DIS nanoparticles and MC (Dis-NPs/ISG) and found that the Dis-NPs/ISG systems are effective ophthalmic DDSs for retinal drug delivery. In addition, we found that the collapse of ATP production due to excess NO caused a decrease in the amplitude of ERGs  in the retinas of 15-month-old OELTF rats and that the repetitive instillation of the Dis-NPs/ISG recovered the deterioration of ERGs by inhibiting the iNOS expression in the retina. These findings provide important information that can be used to design further studies aimed at developing novel therapeutic approaches to DR.


Nd/Sd ratio from the lineage leading to SARS-CoV-2 to the strains of the current pandemic, suggesting that the vast majority of nonsynonymous mutations currently segregating within the human strains have a negative impact on viral fitness. Finally, we estimate that the time to the most recent common ancestor of SARS-CoV-2 and RaTG13 or RmYN02 based on synonymous divergence is 51.71 years  and 37.02 years , respectively.Human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is most closely related, by average genetic distance, to two coronaviruses isolated from bats, RaTG13 and RmYN02. However, there is a segment of high amino acid similarity between human SARS-CoV-2 and a pangolin-isolated strain, GD410721, in the receptor-binding domain (RBD) of the spike protein, a pattern that can be caused by either recombination or by convergent amino acid evolution driven by natural selection. We perform a detailed analysis of the synonymous divergence, which is less likely to be affected by selection than amino acid divergence, between human SARS-CoV-2 and related strains. We show that the synonymous divergence between the bat-derived viruses and SARS-CoV-2 is larger than between GD410721 and SARS-CoV-2 in the RBD, providing strong additional support for the recombination hypothesis. However, the synonymous divergence between pangolin strain and SARS-CoV-2 is also relatively high, which is not consistent with a recent recombination between them, instead, it suggests a recombination into RaTG13. We also find a 14-fold increase in the  RaTG13RS-CoV-2 . And RaTRS-CoV-2 . Furtherd RaTG13 . Xiao etd RaTG13 . Similard RaTG13 . Additiod RaTG13 . They did RaTG13 . Detailed RaTG13 . This wo the RBD . Moreove the RBD a.To analyze the history of these sequences further, we here focus on patterns of synonymous divergence, which has received less focus, but also is less likely to be affected by selection than amino acid divergence. We develop a bias corrected estimator of synonymous divergence specific for SARS-CoV-2 and related strains, and analyze divergence using both sliding windows and a whole-genome approach between SARS-CoV-2 and related viral strains.2.1http://ftp://ftp.ebi.ac.uk/pub/databases/embl/release/std), NCBI Virus Genomes (http://ftp://ftp.ncbi.nlm.nih.gov/genomes/Viruses), NCBI Virus Genbank Entries , NCBI Influenza Genomes (http://ftp://ftp.ncbi.nlm.nih.gov/genomes/INFLUENZA/), all Whole Genome Shotgun (https://www.ncbi.nlm.nih.gov/genbank/wgs/) assemblies under taxonomy ID 10239, along with GISAID Epiflu and EpiCoV databases. Recently published sequences from the Myanmar bat samples  genome. The genome alignments were performed using MAFFT (v7.450)  with par2.3We detected possible recombination events across the genome using a combination of seven algorithms: RDP , GENECON2.4Nd or Sd distance matrices which estimated using codeml (We estimated phylogenetic trees using two methods: neighbor joining (NJ) and maximum likelihood (ML). The NJ trees were estimated using g codeml  with parg codeml . For ML g codeml  (v1.5.2)g codeml  that shog codeml  were: 14g codeml .2.5N/dSd ratio Sd, varied between 0.25 and 3.00. Estimates of Sd > 3 are truncated to 3. For simulations of genome-wide divergence between RaTG13 and human strains, we fix Sd at 0.1609 (the ML estimate outside the RBD region reported in Section 3). In all cases, we use 10,000 independent replicate simulations for each parameter setting.We simulated divergence with realistic parameters for SARS-CoV-2 using a continuous-time Markov chain under the F3x4 codon-based model , which p2.6Nd and Sd were estimated using two different methods implemented in the PAML package ( package  (version package  as imple package  implemen2.7Sd (see Section 3), we identified a quartic function which maps from Sd, into Sd. To identify the coefficients of this function, we used 10,000 simulations as described previously, on a grid of Sd values . We then identified coefficients such that sum of (Sd)2 is minimized over all simulation values.To correct for the biases observed in the estimation of ACE2 contact residues region of the S protein, were the pangolin SARS-CoV-2 virus. These observations are consistent with previous results that RaTG13 and RmYN02 are the most closely related viral strains, while the region containing the ACE2 contact residues is more closely related to the pangolin virus strain . In adds strain  .We focus further on studying the synonymous evolution of SARS-CoV-2, and analyzing Wuhan-Hu-1 as the human nCoV19 reference strain  along wiWe performed recombination analyses across the five viral genomes based on the consensus of the seven recombination-detection methods implemented in RDP5 (see Section 2). We identified nine recombination regions affecting at least one of the sequences . PhylogeS gene where the divergence between SARS-CoV-2 and GD410721 is moderate and the divergences between GD410721 and both SARS-CoV-2 and RaTG13 are quite high and show very similar pattern. This, as also found in the recombination analyses based on methods implemented in RDP5, would suggest a recombination event from a strain related to GD410721 into an ancestor of the human strain  between SARS-CoV-2 and the four other strains analyzed in windows of 300 bp . Notice n strain , or alten strain . We noten strain . The othn strain , suggestn strain , suggestS gene encoding the spike protein could suggest the presence of recombination in the region, previous study (Sd) in sliding windows of 300 bp. However, estimation of Sd is complicated by the high levels of divergence and extremely skewed nucleotide content in the third position of the sequences  for evaluating the performance of the ML estimator of Nd and Sd (as implemented in codeml (Sd < 1.5 (N/dSd ratio observed for SARS-CoV-2 (see Section 2). The bias corrections we obtain are Sd >2, the estimates are generally not reliable, however, we note that for Sd < 1.5 the bias-corrected ML estimator tends overall to have slightly lower MSE, and we, therefore, use this estimator for analyses of 300 bp regions.While the overall divergence in the us study  reportedequences  which wiequences . It is sN and dS . Howevern codeml ) under tdS < 1.5 . Howevervariance  so that Nd and Sd under the F3x4 model in codeml  between SARS-CoV-2 and RaTG13 and Sd = 0.2043, Nd = 0.0220 (N/dSd = 0.1077) between SARS-CoV-2 and RmYN02. However, a substantial amount of this divergence might be caused by recombination with more divergent strains. We, therefore, also estimate Nd and Sd for the regions with inferred recombination tracts  and Sd = 0.1117  between SARS-CoV-2 and RaTG13 and RmYN02, respectively. This confirms that RmYN02 is the virus most closely related to SARS-CoV-2. The relative high synonymous divergence also shows that the apparent high nucleotide similarity between SARS-CoV-2 and the bat strains (96.2% (N/dSd = 0.0410 and 0.0555) exacerbated by a high degree of synonymous homoplasy facilitated by a highly skewed nucleotide composition at the third position of codons  is causSd will be larger than 1, and with this level of saturation, estimation of divergence is associated with high variance and may be highly dependent on the accuracy of the model assumptions. This makes phylogenetic analyses based on synonymous mutations unreliable when applied to these more divergent sequences. Nonetheless, the synonymous divergence levels seem generally quite compatible with a molecular clock with a Sd of 0.9974 , 1.0366 , 1.0333 , and 1.0304  between the outgroup, GX_P1E, and the three ingroup strains. The largest value is observed for RaTG13 (Sd = 1.0366), despite this sequence being the earliest sampled sequence, perhaps caused by additional undetected recombination into RaTG13.The synonymous divergence to the pangolin sequences GD410721 and GX_P1E in genomic regions with inferred recombination tracts removed is 0.5095  and 1.0304 , respectively. Values for other comparisons are shown in Sd between SARS-CoV-2, GD410721, and RaTG13 and the ratio of Sd  to Sd  in 300 bp sliding windows along the genome. Notice that we truncate the estimate of Sd at 3.0. Differences between estimates larger than 2.0 should not be interpreted strongly, as these estimates have high variance and likely will be quite sensitive to the specifics of the model assumptions.To address the issue of possible recombination, we plot Sd  approximately equals Sd  and is larger than Sd  in almost the entire genome showing than in these parts of the genome GD410721 is a proper outgroup to  assuming a constant molecular clock. One noticeable exception from this is the RBD region of the S gene. In this region, the divergence between SARS-CoV-2 and GD410721 is substantially lower than between GD410721 and RaTG13  to Sd   are saturated in this region, even though they are substantially lower than 1 in the rest of the genome. This strongly suggests a recombination event in the region and provides independent evidence of that previously reported based on amino acid divergence (e.g. We find that d RaTG13 . The samd RaTG13 . The pat RaTG13) . In factnce e.g. .ACE2 contact residue region of the spike protein, which is more consistent with the hypothesis of recombination into RaTG13, as proposed in The combined evidences from synonymous divergence and the topological recombination inference provide strong support for the recombination hypothesis. However, these analyses alone do not distinguish between recombination into RaTG13 from an unknown source as previously hypothesized  and recoACE2 contact residue region supports the clustering of SARS-CoV-2 and GD410721, but with unusual long external branches for all strains except SARS-CoV-2, possibly reflecting smaller recombination regions within the ACE2 contact residue region.Unfortunately, because of the high level of synonymous divergence to the nearest outgroup, tree estimation in small windows is extremely labile in this region. In fact, synonymous divergence appears fully saturated in comparison with GX_P1E, eliminating the possibility to infer meaningful trees based on synonymous divergence. However, we can use the overall ML tree using both synonymous and nonsynonymous mutations . The ML Nd/Sd ratio for longer evolutionary lineages. Furthermore, changes in effective population size will translate into changes in the rate of substitution of slightly deleterious mutations. Finally, changes in ecology  can lead to changes in the rate of amino acid substitution. For all of these reasons, the use of synonymous mutations, which are less likely to be the subject of selection than nonsynonymous mutations are preferred in molecular clock calculations. For many viruses, the use of synonymous mutations to calibrate divergence times is not possible, as synonymous sites are fully saturated even at short divergence times. However, for the comparisons between SARS-CoV-2 and RaTG13, and SARS-CoV-2 and RmYN02, synonymous sites are not saturated and can be used for calibration. We find an estimate of ω  =  0.0391 between SARS-CoV-2 and RaTG13, excluding just the small RDB region showing a recombination signal in SARS-CoV-2  of −3 substitutions/site/year . The synonymous specific mutation rate can be found from this as Sd/year = pS  ωpN), where ω is the N/dSd ratio, and pN and pS are the proportions of nonsynonymous and synonymous sites, respectively. The estimate of the total divergence on the two lineages is then Sd is small and the uncertainty is mostly dominated by the uncertainty in the estimate of the mutation rate. We estimate the SD in −3 and SD 0.18 −3, and mean 0.5604 and SD 0.1122, respectively. We subject each simulated data set to the same inference procedure as done on the real data. Our estimate of the SD in the estimate is 11.8 for RaTG13 versus SARS-CoV-2 and 9.41 for RmYN02 versus SARS-CoV-2, providing an approximate 95 per cent CI of  and , respectively. For RaTG13, if including all sites, except the 244 bp in the RBD of the S gene  and 37.02 years , respectively. Most of the uncertainty in these estimates comes from uncertainty in the estimate of the mutation rate reported for SARS-CoV-2. As more data are being produced for SARS-CoV-2, the estimate should become more precise and the CI significantly narrowed. We note that the mutation rate we use here are estimated based on the entire genome, which may differ from that in nonrecombination regions. To address this problem, we downloaded all the SARS-CoV-2 sequences that are available until 17 August 2020 from GISAID, and obtained an estimate of 1:0.81 for the ratio of mutation rates in the recombination and nonrecombination regions, using the ‘GTRGAMMA’ model implemented in the RAxML (Stamatakis 2014). Given the length ratio between the two partitions is 1:4, the difference between the partitions will cause a slight overestimate of the mutation rate by ∼5 per cent, which is relatively small compared to the confidence intervals and the potential for other unknown sources of uncertainty. However, we warn that a residual cause of unmodeled statistical uncertainty is deviations from the molecular clock. Variation in the molecular clock could be modeled statistically . The accession codes for nonhuman sequences can be found in Virus Evolution online.veaa098_Supplementary_DataClick here for additional data file.


Vitiligo is a common skin disorder in which melanocytes are destroyed by auto-reactive immune responses. The loss of melanocytes results in the appearance of depigmented areas in different parts of the body. Cytokines have remarkable roles in the pathogenesis of vitiligo, such as IL-1, IL-6, and TNF-α; interleukin 27 (IL-27) is a new member of the IL-6/IL-12 family, mainly released by activated antigen-presenting cells. IL-27 has been suggested to function as a pro-inflammatory as well as an anti-inflammatory cytokine. Altered concentrations of IL-27 have been shown in various auto-immune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. No studies have been conducted to determine the expression of this cytokine in vitiligo patients.The objective of this study was to determine the serum concentration of IL-27 in vitiligo patients and compare it with normal individuals.The serum concentration of IL-27 in 79 vitiligo patients was evaluated in comparison to 45 healthy controls using ELISA assay.p = 0.026). Furthermore, no correlation between IL-27 concentrations and disease parameters such as vitiligo severity and the extension of the depigmented area was observed.Results showed decreased concentration of IL-27 in vitiligo patients as compared with healthy subjects (A larger sample size would be more recommended for this study.The reduction in the serum levels of IL-27 in vitiligo patients compared to normal subjects suggested the possible anti-inflammatory role of this cytokine in vitiligo. Thus, IL-27 may be considered as a new target for the manipulation of the immune system in vitiligo patients. Vitiligo is a common acquired depigmentation disorder characterized by white patches which have been distributed in several parts of the body and greatly affects the quality of life and mental health of the patients.The main causes of vitiligo are complex and maybe stem from genetic disorders, which can feature polygenic and multifactorial inheritance.13IL-27, a rather new member of the IL-6/IL-12 family, is mainly released by activated antigen-presenting cells, including dendritic cells (DCs), monocytes, and macrophages.Although previous studies identified the inflammatory role of IL-27, the latest evidence suggests an immunomodulatory effect of this cytokine.Regarding the complex pro-inflammatory and anti-inflammatory nature of IL-27, several studies have investigated the local or systemic concentrations of IL-27 in different diseases. Alteration in the concentration of IL-27 and its correlation with autoimmune parameters have also been reported in several Th1/Th17-mediated inflammatory disorders, such as multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, and rheumatoid arthritis.36The concentrations of IL-27 in vitiligo patients have not yet been investigated. The present examination intended to evaluate the serum levels of IL-27 in vitiligo patients.Seventy-nine vitiligo patients including 32 (41%) males and 47 (59%) females, and 45 age and sex-matched healthy controls with no sign of autoimmune diseases were enrolled in the study. The study population included 72 patients with generalized vitiligo and seven cases with localized vitiligo. Participants in this study assented to join according to the ethics committee of the medical school of Shiraz University of Medical Sciences (IR.sums.med.rec.1397.400).From all study populations, 5 mL of peripheral blood was collected. After centrifugation at 3000 rpm for 10 min, the sera were separated and kept at −70 °C until used.Serum concentrations of IL-27 were distinguished by enzyme-linked immunosorbent assay (ELISA) in vitiligo patients and healthy subjects. IL-27 concentrations were quantified using an ELISA kit  manufacturers’ instructions. The sensitivity of tests was 12.8 pg/mL. Briefly, 100 μL of capture antibody was coated in each well of a 96-well microplate for overnight at room temperature (RT). After washing and blocking, 100 μL serum samples of patients and controls were added for 2 h, at RT. Plates were washed again and 100 μL of the detection antibody and then 100 μL of the working dilution of streptavidin horseradish peroxidase was added for 20 min. Then substrate solution (100 μL) was added for 20 min; finally, the reaction was stopped by adding stop solution to each well. The optical density of samples was read using a microplate reader at 450 nm. The levels of cytokine were extrapolated from the related standard curve.U and Kruskal–Wallis) tests were used; p-values < 0.05 were considered as significant. Graphs were constructed using Graph Pad Prism . All data are presented as mean ± standard error of the mean (SEM), unless otherwise specified.All data were analyzed by SPSS v. 16 software  and according to the normality test (Kolmogorov-Smirnov test); non-parametric .The serum concentrations of IL-27 in 79 patients with generalized (72 cases) or localized (seven cases) vitiligo were observed using the ELISA technique. Forty-five age and sex-matched healthy subjects were used as the control group. As presented in The correlations between serum concentrations of IL-27 with gender, the severity of the disease, two types of  vitiligo, segmental/nonsegmental forms of the disease, and response to treatment in patients with vitiligo were examined. There was no significant correlation between IL-27 levels and these parameters in the present study .Table 2CThe main reason for vitiligo is ill defined. Human and experimental studies have recently provided considerable evidence regarding the pattern of autoimmunity in the pathogenesis of vitiligo. Both humoral and cellular immunity have shown to be involved in the etiology of the disease.41IL-27 has been suggested to function as a pro-inflammatory as well as an anti-inflammatory cytokine. Altered concentrations of IL-27 have been shown in several autoimmune and skin disorders. Despite early studies identified pro-inflammatory role of IL-27, latest evidence suggests that it suppresses a range of immune cell proliferation and cytokine production.46Based on those explanations and the results of several studies about the altered expression of IL-27 in autoimmunity and skin disorders, it was hypothesized that the concentration of IL-27 in vitiligo patients might be altered. Consistent with previous studies in SLE and BD patients, the present study showed that the concentrations of IL-27 serum levels were reduced in vitiligo patients, which can be justified through some probable mechanisms. First, IL-27 acts against Th17 development in a direct manner through modulating DCs and induces IL-10 production by naïve CD4+ T-cells.It can be concluded that IL-27 has an immunomodulatory role in vitiligo. According to the observations of the present study, there was no association between IL-27 serum level and demographic information, severity of the disease, types of vitiligo, and response to treatment in patients with vitiligo. Further studies with more patients of all types of vitiligo are needed to reveal the possible alterations in the concentration of IL-27 in those patients and their correlation with disease characteristics, including severity and the extension of the affected skin area. In conclusion, IL-27 has two roles; it should be considered as a new target for the manipulation of the immune system in various immune-mediated disorders.10.13039/501100004320Shiraz University of Medical Sciences (grant No. 17684).This study was supported by Saeed Malek Hosseini: Drafting and editing of the manuscript.Naser Gholijani: Collection, analysis, and interpretation of data; participation in the study design.Nooshafarin Chenari: Collection, analysis, and interpretation of data; participation in the study design.Nooshafarin Chenari: Drafting and editing of the manuscript.None declared.


These results strongly support the conclusion that high levels of MCL-1 expression enable the oncogenesis of PEL cells and thus, MCL-1 could be a potential drug target for KSHV-associated PEL. This work also unravels a mechanism by which an oncogenic virus perturbs a key component of the ubiquitination pathway to induce tumorigenesis.Primary effusion lymphoma (PEL) is an aggressive B cell lymphoma that is etiologically linked to Kaposi’s sarcoma-associated herpesvirus (KSHV). Despite standard multi-chemotherapy treatment, PEL continues to cause high mortality. Thus, new strategies to control PEL are needed urgently. Here, we show that a phosphodegron motif within the KSHV protein, latency-associated nuclear antigen (LANA), specifically interacts with E3 ubiquitin ligase FBW7, thereby competitively inhibiting the binding of the anti-apoptotic protein MCL-1 to FBW7. Consequently, LANA-FBW7 interaction enhances the stability of MCL-1 by preventing its proteasome-mediated degradation, which inhibits caspase-3-mediated apoptosis in PEL cells. Importantly, MCL-1 inhibitors markedly suppress colony formation on soft agar and tumor growth of KSHV + PEL.Primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma, is associated with Kaposi’s sarcoma-associated herpesvirus (KSHV). However, the underlying mechanisms that govern the aggressiveness of KSHV-associated PEL are poorly understood. Here, we demonstrate that KSHV LANA interacts with cellular ubiquitin E3 ligase FBW7, sequestering MCL-1 from FBW7, which reduces MCL-1 ubiquitination. As such, LANA potently stabilizes and increases MCL-1 protein, leading to inhibition of caspase-3-mediated apoptosis in PEL cells. Furthermore, MCL-1 inhibitors efficiently blocked PEL progression in mouse xenograft model. These results suggest that LANA acts as a proto-oncogene via deregulating tumor suppressor FBW7, which upregulates anti-apoptotic MCL-1 expression. This study suggests drugs that target MCL-1 may serve as an effective therapy against KSHV The ubiquitin-proteasome system (UPS) is a network of proteins involved in ubiquitination of cellular targets and subsequent control of numerous cellular functions. Dysregulation of the components of this elaborate network leads to human diseases such as cancer . One prePEL, caused by Kaposi’s sarcoma-associated herpesvirus (KSHV), is an aggressive B cell malignancy that occurs in peritoneal spaces and body cavities  –14. The Here, we identified the mechanism by which KSHV drives high expression of MCL-1 by targeting E3 ubiquitin ligase FBW7 and clarified its role in KSHV-associated lymphoma. We found that KSHV LANA interacts with FBW7 in a phosphodegron motif-dependent manner and that this interaction specifically suppresses MCL-1 ubiquitination, eventually inhibiting caspase-3-dependent apoptosis. Furthermore, depletion of MCL-1 by small interfering RNA (siRNA) or pharmacological inhibition of MCL-1 using the small-molecule inhibitor AT-101  markedlyFig 1A) that appear to be recognized by FBW7. To validate whether LANA interacts with FBW7 through its CPD motifs, we first carried out immunoprecipitation (IP) experiments to examine the interaction between LANA and FBW7. Notably, we found that LANA interacted with FBW7  but not with other F-box family proteins . Since substrate phosphorylation is critical for being recognized by FBW7 . We. WeFig 56 clones . Accordi6 clones , suggest6 clones . We foun+ BCBL-1, BC-3 cells, and KSHV+EBV+ BC-1 cells than in KSHV− BJAB cells . Consistent with this, endogenous MCL-1 ubiquitination was significantly lower in BCBL-1 cells than in BJAB cells upon MG132 treatment, indicating that high levels of MCL-1 protein correlates inversely with ubiquitination of endogenous MCL-1 . In addition, we further verified that LANA competes with MCL-1 for FBW7 binding via endogenous FBW7 IP in BCBL-1 cells , followed by crystal violet staining to count colonies. Notably, both co-treatment of BCBL-1 cells with MCL-1 inhibitors and treatment of cells with MCL-1 inhibitors after colony formation led to marked inhibition of colony formation . Given that abdominal distention reflects the development of ascites, we collected and measured the volume of ascites produced in each mouse. On average, 1.77 ml ascites were collected from DMSO-treated mice versus 0.12 ml from AT-101-treated mice . The volume of ascites correlated with the increase in body weight , verifying that weight gain was attributable to tumor establishment. Previous studies reported that mice injected with KSHV+ PEL cells, including BCBL-1 cells, exhibited notable splenomegaly when compared with normal NOD/SCID mice due to infiltration of anaplastic cells  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. 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Please don't hesitate to contact us if you have any questions or comments.Sincerely,Pinghui FengAssociate EditorPLOS PathogensErik FlemingtonSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Myeloid cell leukemia 1 (MCL-1) is an anti-apoptotic protein that is critical for survival of immune cells including T lymphocytes, B lymphocytes, and macrophages. Recently, MCL-1 has emerged also as an essential factor for the survival of KSHV-infected primary effusion lymphoma (PEL) cell lines. Here, the paper re-invited the essential role of MCL-1 in KSHV infection using MCL-1 depletion or inhibitors in cultured PEL cell lines and a xenograft mouse model of PEL. As an original part of the manuscript, the authors surveyed the molecular mechanism by which MCL-1 is stabilized in the PEL cell lines and proposes that LANA competes with MCL-1 for binding to the ubiquitin E3 ligase FBW7, thus reducing ubiquitination and degradation of MCL-1. This proposed model is supported by the use of the LANA mutant P1, identified by the authors, which is incapable of binding to FBW7. While the novelty of the paper is mainly represented by the molecular mechanism of action for LANA-mediated MCL-1 stabilization, key pieces of evidence are still missing, in particular, in the context of virus infection, and the results of polyubiquitination of MCL-1 is not convincing. In addition, the paper lacks necessary discussion on already published LANA interaction with FBW7 and on how the mitochondrial protein MCL-1 is regulated by the nuclear proteins LANA and FBW7.Reviewer #2: In this interesting manuscript, Kim et al. describe a novel mechanism of how KSHV LANA stabilises MCL-1, an anti-apoptotic cellular protein, which has previously been shown by others to be highly expressed in and to be essential for the survival of primary effusion lymphoma (PEL) cells and to be a 'druggable' target to treat PEL in preclinical models. The manuscript by Kim et al. therefore contributes two important aspects to the KSHV literature: (i) it shows that KSHV LANA stabilises MCL-1 by binding to and thereby diverting the E3 ligase FBW7, which normally downregulates MCL-1; (ii) it describes two further MCL-1 or FBW-7 inhibitors that potently inhibit PEL growth in an in vivo preclinical model and thereby extends a previous report on another MCL-1 inhibitor, S63845, which had been shown by others to markedly inhibit the growth of of PEL cells in tissue culture. The experiments described in this very well written manuscript are of high technical quality.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: 1. LANA-mediated MCL-1 stabilization and related mechanisms were proposed mainly based on the studies using sole expression of LANA in KSHV-negative BJAB cell line and 293T cells. So, it is not appreciated how important LANA, in particular, via sequestering FBW7, contributes to the elevated expression of MCL-1 in KSHV-infected PEL cell lines as shown in Figure 5A. To address this point, it is desirable to use recombinant KSHV viruses, e.g., BAC16, harboring LANA wild-type (WT), knockout, and revertant with WT or the P1 mutant, for MCL-1 assays in BJAB cells. If applicable, it’d be also nice to identify and use a competitive inhibitory peptide that can block the interaction between LANA and FBW7 in KSHV-infected PEL cells.2. The paper examined protein interactions using immunoprecipitation of ‘overexpressed’ proteins. Despite the presence of solid evidence, the interactions among LANA, FBW7, and MCL-1 should be further investigated in terms of biological relevance, e.g., i) following etoposide treatment in the BJAB cells expressing LANA WT or P1 and ii) in the PEL cell lines along with control cell BJAB. In either case, MG132 treatment may be needed to preserve MCL-1 protein to make the level of MCL-1 comparable as shown Figure 5B.3. It is not convincing that the ubiquitinated bands shown in Figures 3B and 5B represent polyubiquitination of ‘MCL-1’. The paper did not describe how MCL-1-associated proteins, probably also ubiquitinated, were removed from the MCL-1-immunoprecipitated complex in the Materials and Methods section. Sonication could not remove MCL-1 interactions with other cellular proteins. In addition, in Figure 3B, controls are missing such as transfection of MCL-1 and HA-Ub or HA-Ub alone.An increase on polyubiquitination of MCL-1 does not necessarily implicate enhanced proteasomal degradation of MCL-1. Thus, it is desirable to identify what type of polyubiquitination, e.g., K48 and K63, of MCL-1 in the Figures and to examine whether LANA can suppress K48-linked polyubiquitination, which is involved in proteasomal degradation, of MCL-1.4. In fact, the interaction of LANA and FBW7  was previously reported by Lan K, et al. in PNAS (2007), who demonstrated that the oncogenic factor intracellular activated notch (ICN) could be stabilized by LANA competition with FBW7. ICN is a reminiscence of MCL-1. However, both studies show the discrepant results in FBW7-binding regions in LANA. It needs to be addressed. It is curious why ICN was not involved in Figure 2B.Reviewer #2: no major criticisms. In my opinion, no additional experiments are needed to support the conclusions drawn in this manuscript.**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: 1. MCL-1 is mainly detected and functional in mitochondria. How can the nuclear proteins LANA and FBW7  stabilize the mitochondrial protein MCL-1?2. It is desirable to show the levels of primary (target) proteins, in addition to binding proteins, immunoprecipitated in Figures 1B, 1C, 1D, 1E, 3A, 3B, and 5B.3. In the top panels of Figures 3B and 5B, how do the authors know that the bottom bands are ‘unmodified MCL-1’? It is unlikely to detect the MCL-1 bands with anti-HA or ubiquitin antibody. Is it possible that the bands are the heavy chain of IgG used in immunoprecipitation?4. In lines 228-229, the statement, “LANA enhanced endogenous MCL-1 protein levels ”, should be rephrased; for example, LANA protected MCL-1 from etoposide-induced degradation. However, ratios of the MCL-1 proteins before and after etoposide treatment look similar among vector, LANA, and LANA-P1 cells. Thus, it may be unlikely that LANA protected MCL-1 from etoposide-induced degradation. It’d be helpful to show the relative band intensities of MCL-1 of each etoposide-treated cell compared to no etoposide treatment.5. It’d be nice to show the levels of FBW7 in BJAB and the PEL cell lines in Figure 5B.6. How many mice were used in each group of the xenograft experiment? In Figure 7B, does the number of dots in ascites volume represent the number of sacrificed mice? Detailed description and statistic justification of the animal experiments should be addressed.Reviewer #2: 1. Fig. 1F, text line 161: co-localisation of LANAwt and LANA P1 with FBW7: if LANA is transfected into cells in the absence of the KSHV genome, it shows a diffuse nuclear distribution which does not lend itself to co-localisation studies. The experiment shown in fig. 1F therefore only shows that the LANA P1 mutant localises to the nucleus, not that it 'co-localises' with FBW7 (line 161 in the text). I assume that the authors may only have wanted to make the point that the LANA P1 mutant has a nuclear localisation. They should therefore change the wording in the text on line 161.2. Fig. 5C bottom panel: the WB for BJAB cells suggests that MCL-1 expression levels in BJAB are similar to those in the three PEL cell lines, in contrast to Fig. 5A, which indicates that MCL-1 levels are higher in PEL cells than in BJAB . Presumably this is due to a longer exposure of the WB for BJAB in Fig. 5C. Comparable exposure times should be used for the WBs shown in figure 5C.3. Fig. 5D, text line 256: the text states that AT101 induced cell death in PEL cells but not in BJAB; according to fig. 5D, there is a small increase in the number of dead cells also in BJAB cells. AT1210477 also reduces the growth rate of BJAB in fig. S2D, albeit not to the same extent as in PEL cells. The corresponding statements in the text should be rephrased.4. line 306: "one of attempt" - better: "an attempt"**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here on PLOS Biology: Reproducibility:http://journals.plos.org/plospathogens/s/submission-guidelines#loc-materials-and-methodsTo enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see AttachmentComments for the authors.docxSubmitted filename: Click here for additional data file. 5 Nov 2020AttachmentResponse to Reviewers-final.docxSubmitted filename: Click here for additional data file. 22 Nov 2020Dear Hey-Ra,We are pleased to inform you that your manuscript 'Kaposi’s Sarcoma-associated Herpesvirus Latency-associated Nuclear Antigen Dysregulates Expression of MCL-1 by Targeting FBW7' has been provisionally accepted for publication in PLOS Pathogens. As you can see from reviewer 1, there are some minor points that you will need to address. Please revise accordingly. Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Pinghui FengAssociate EditorPLOS PathogensErik FlemingtonSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: Kim et al present a revised manuscript that is considerably strengthened in its demonstration of LANA regulation of MCL-1 in the context of virus-infected cells. The authors have enthusiastically performed ubiquitination assays in response to my comments.Reviewer #2: Kim et al. describe a novel mechanism of how KSHV LANA stabilizes MCL-1, an anti-apoptotic cellular protein, which has previously been shown by others to be highly expressed in and to be essential for the survival of primary effusion lymphoma (PEL) cells and to be a 'druggable' target to treat PEL in preclinical models. The manuscript by Kim et al. therefore contributes two important aspects to the KSHV literature: (i) it shows that KSHV LANA stabilizes MCL-1 by binding to and thereby diverting the E3 ligase FBW7, which normally downregulates MCL-1; (ii) it describes two further MCL-1 or FBW-7 inhibitors that potently inhibit PEL growth in an in vivo preclinical model and thereby extends a previous report on another MCL- 1 inhibitor, S63845, which had been shown by others to markedly inhibit the growth of PEL cells in tissue culture.In this revised version of their manuscript, the authors have carefully addressed all the reviewers' suggestions. The manuscript is very well written and the reported experiments are of high technical quality.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: NoneReviewer #2: No further experiments required**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: The authors did not quite understand the concern (minor Q2) raised on co-immunoprecipitation (co-IP) assays. The comment was to show the amounts of primary (target) proteins immunoprecipitated in Figures 1B, 1C, 1D, 1E, 3A, 3B, and 5B. The level of “co-IPed protein” relies on the amount of protein targeted primarily for immunoprecipitation. Thus, in addition to the input (lysate), the levels of the immunoprecipitated primary proteins should be shown; for example, in Fig. 1E, immunoprecipitated Flag-FBW7 should be presented by immunoblotting with anti-Flag antibody.Reviewer #2: no further minor points identified**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript, if you opted to have an early version of your article, will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Pathogens. Best regards,Kasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064


The Perceived Stress Scale (PSS) is a globally used and self-report scale measuring perceived stress. Three versions of PSS  are available which comprise 14, 10 and 4 items respectively. However, the Chinese version of the PSS has not yet been validated in a large community-based general population. The aims of this study were to evaluate the psychometric properties of the Chinese PSS in a large community-based general population and to compare the appropriateness of the three versions of PSS.A total of 9507 adults with at least a junior high school education and completed PSS-14 from the China Health and Nutrition Survey were involved in this study. The internal consistency reliability of PSS was assessed using Cronbach’s alpha coefficient and confirmatory factor analysis was employed to test the construct validity. Modification index was used for model extension and the critical ratio was used for model restriction.The internal consistency coefficients were satisfactory for PSS-14 and PSS-10, but not for PSS-4. The corresponding Cronbach’s alpha were 0.830, 0.754 and 0.473 respectively. A 2-factor structure was confirmed for the PSS-14 and PSS-10, and all items’ standardized factor loadings exceeded 0.4 for either negative or positive factors. Given that item 12 loaded on both negative and positive factors for PSS-14 and the goodness of fit for PSS-14 was not acceptable, PSS-13 (PSS-14 excluding item 12) was studied. The construct validities of PSS-13 and PSS-10 were satisfactory, but the goodness of fit for PSS-10 were better than that for PSS-13.PSS-13 (PSS-14 excluding item 12) and PSS-10 have satisfactory psychometric properties. PSS-10 are more applicable to measure the perceived stress than PSS-13 in a large community-based general population in China. The concept of stress can be classified approximately into three perspectives which are environmental, psychological and biological stress . PreviouThe PSS has been translated into many languages and the reliability and validity have been verified in different countries. For instance, PSS-14 has been evaluated in Sweden , France The participants were from the China Health and Nutrition Survey co-operated by the National Institute for Nutrition and Health of the Chinese Center for Disease Control and Prevention and the University of North Carolina at Chapel Hill in the United States . The ongThe PSS was designed for use with community samples with at least a junior high school education . Therefo. The negative subscale negatively states items, and is intended to assess lack of control and negative reactions (perceived distress), while the positive subscale positively states items, and measures the degree of ability to cope with existing stressors (coping capacity) [The original PSS consists of 14 items (PSS-14) which was translated from English into Chinese, and subsequently back into English to ensure the accuracy of translation. Each item is rated on a 5-point Likert-type scale, ranging from 0 = ‘never’ to 4 = ‘very often’. The scale can cluster into two subscales: negative subscale  and positive subscale apacity) . PSS-10,The internal consistency reliability of the three versions of PSS was examined by Cronbach’s alpha and the reasonable acceptability criterion of which is ≥0.70.P value to ensure the results’ completeness.The construct validity was examined by confirmatory factor analysis (CFA). Using the generalized least squares method, the two-factor models were fitted for different versions of PSS respectively to assess the goodness-of-fit of the factor structure. Models with goodness-of-fit index (GFI) > 0.9, adjust goodness-of-fit index (AGFI) > 0.9, comparative fit index (CFI) > 0.9, standardized root mean square residual (SRMR) < 0.08, and root mean square error of approximation (RMSEA) < 0.08 were regarded as a good fit. Little suggested that investigators ought not rely too heavily on chi-square test for comparing competing models, but rather on the indices mentioned above to determine the overall adequacy of a fitted model, for the chi-square value was an overly sensitive index of fit when working with large measurement models. Therefore, we reported chi-square value, freedom degree and corresponding p < 0.05.The CFA analyses were performed by Amos 24.0. Cronbach’s alpha was obtained using SPSS 21.0. All statistical tests were two-tailed and employed a significance level at Two kinds of model modification indices were used, of which the modification index(M.I.) was used for model extension and the critical ratio(C.R.) was used for model restriction. Modification priority was given to the path with the maximum M.I. value or C.R. value.The sample consisted of 9507 individuals with a mean age of 47.5 years SD = 14.1) and 51.1% of the sample were men. The majority (88.4%) of the participants were married. The demographics are presented in Table 4.1 and 5The Cronbach’s alpha was 0.830  for the PSS-14 and 0.754  for the PSS-10. When each item of the PSS − 14 and PSS-10 was deleted from the analysis in order to test the robustness, Cronbach’s alpha remained high (0.811–0.829 for the PSS-14 and 0.728–0.739 for the PSS-10). The Cronbach’s alpha was 0.473 for the PSS-4. When each item of the PSS − 4 was excluded, Cronbach’s alpha ranged from 0.295 to 0.495.The goodness-of-fit indices of confirmatory factor analysis Table  presenteP = 0.819).Table P = 0.169). The mean score significantly decreased with age from 19.6 in the 18–44 age group to 18.6 in the 60–94 age group (P < 0.001). In addition, the PSS-10 score of participants who were employed was the highest.Table This study verified the reliability and construct validity of the Chinese version of the Perceived Stress Scale . To our knowledge, this study is the first to evaluate the psychometric properties of the PSS in a large general community-based population in China. The results presented that the PSS-10 and modified PSS-14 were suitable for this population, while PSS-14 and PSS-4 did not have adequate psychometric properties.Cronbach’s alpha values in this current study revealed that not only the overall PSS-14 and PSS-10, but also each of the two subscales of PSS-14 and PSS-10 were internally reliable, but PSS-4 was not. These findings were in line with previous studies in different countries, such as China , Japan , Korea , Thailans = 0.120), and were not correlated for PSS-10, we believed that it was acceptable no matter using the sub-scales as a whole or using them separately.Our study supported a two-factor structure of the 14-, 13- and 10-item versions of PSS which was confirmed by most previous studies , 28, 29.By confirmatory factor analysis, our study found covariance between error terms of items 4 and 5 indicating a systematic error in the response. The existence of error covariance may be due to the high degree of overlap in item content, but it was unclear. However, it was seemingly unlikely due to the subjects’ misunderstanding of item 4  and item 5, because “irritating life hassles” was totally different from “important changes”. More studies were need to analyze the error covariance.Although the psychometric properties of both PSS-13 and PSS-10 were satisfactory, the reliability and validity of PSS-10 were the best when compared to PSS-13. Moreover, it was critical to complete the questionnaire in a shorter time in a large survey with abundant multiple measures. Therefore, we recommended measurement of perceived stress utilizing the PSS-10 among the community-based general population in China.The first advantage of this study is our utilization of a large community-based general Chinese population. The second advantage is that we excluded those with education lower than junior high school, to whom the PSS is not applicable. To our knowledge, no authors have mentioned this in their manuscripts. There are a few limitations. First, the China Nutrition Transition Cohort Study does not involve other psychological investigation. We can only verify the structure validity of PSS, but cannot verify the concurrent validity or other validity.Comprehensively, the results of our study reveal that PSS-13 (PSS-14 excluding item 12) and PSS-10 have satisfactory psychometric properties. PSS-10 are more applicable to measure the perceived stress than PSS-13 in a large community-based general population in China.